February 14, 2020

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The importance of a well-crafted multimethod research project

Neuroscientists benefit from equipment allowing to measure and manipulate neuronal networks and activity with increasing temporal and spatial resolution. While the development of new technologies for neuroscience has been abundant over the last decades, three fields have been at the centre: optical stimulation (via optogenetics), imaging and electrophysiology. The aim of recording deeper in the brain and with higher precision is driving the outbreak of various technologies; making once confidential methods available to a growing number of labs.

In vitro techniques such as patch-clamp recordings or single-cell optogenetic stimulation have made their way out of the traditional electrophysiology workstation to embark on freely moving animals. This multiplication of methods offered scientists the opportunity to diversify their approach to a specific scientific question. By combining brain slices and in vivo techniques for example, researchers can break down neural activity while confirming the behavioural relevance of the finding.

By combining optical and electrophysiology methods both in vivo and in vitro, this paper is representative of the multitude of tools currently available for research groups.

A brief look into the scientific importance of this study

The Izawa et al. project focuses on the link between melanin-concentrating hormone (MCH) neurons and the mechanisms of memory selection during sleep. MCH is a neuropeptide present in the hypothalamus of vertebrates. MCH neurons play an intrinsic part in the regulation of feeding behaviour and sleep-wake cycles (2). Nonetheless, the relationship between sleep and memory is debated. The underlying mechanisms of retention or discarding of memories during sleep are not well understood.

However, it is commonly accepted that the hippocampus plays an important role in the formation and consolidation of memories. Therefore, Dr Yamanaka’s team decided to study the impact of hypothalamic MCH neurons activity on the hippocampus-dependent memory processes during the REM (Rapid-Eye Movement) sleep phase.

What methods did Izawa et al. use to gather information and data (and what equipment is available to perform this type of experiment)?

Izawa et al. combined a variety of methods to study the different aspects of the MCH neurons involvement in memory retention mechanisms. The following sections will describe the technical aspects of these methods as well as the reasons for choosing them. Finally, we will draw a parallel between these methods and the solutions available in our MCI product range.

1. Retrograde tracing

What is retrograde tracing? Retrograde tracing allows for the identification of neurons projecting to a specific region of interest. This technique consists of injecting a tracer in the region of interest. The tracer is then taken up by synaptic terminals and is conveyed by axonal transport towards the cell bodies. After leaving some time for the tracer to reach the cell bodies, the brain is sectioned. Finally, the tracer is localised through fluorescence microscopy.

Why use retrograde tracing?The team wanted to determine whether MCH neurons project to hippocampal neurons. Injecting retrogradely transported beads, coupled with immunohistochemistry, allowed them to determine which neural population projected to the hippocampus.

What technology does MCI recommend?The tracers used in this study are called RetroBeads™. They are microspheres for retrograde tracing that are used in a variety of studies. You can find them on the manufacturer (Lumafluor) website.

2. Neural activation and inhibition using chemogenetic manipulation

What is chemogenetics? Chemogenetics is a technique to manipulate neural activity. It is powerful for experiments requiring long-term, but minimally invasive control.

G protein-coupled receptors are expressed in the neuronal population of interest. They are specifically designed to have low affinity for their native ligand. However, these designer receptors exclusively activated by designer drugs (DREADDs) have high affinity for a synthetic ligand – a ligand otherwise biologically inactive. DREADDs are genetically inserted into a specific neuronal population. Then, systemic administration of the designer drug allows the remote control of the neuronal population of interest, either by activating or inhibiting neuronal firing.

Why use chemogenetics? Chemogenetics is a powerful tool to probe neural circuits while the animal is freely moving. The advantage of chemogenetics, in comparison with optogenetics (described further down), is that no tech is required to be implanted on the rodent. The designer drug is injected at the beginning of the behavioural assay (usually intraperitoneally), with no further interaction or constraint on the animal behaviour.

In this project, the team used chemogenetics to control the activity of MCH neurons. Combined with various behavioural assays, they could thus evaluate the implication of MCH neurons in memory processes.

What technology does MCI recommend? An informative resource for choosing your chemogenetic technology is the eBook “Practical Considerations for the Use of DREADD and Other Chemogenetic Receptors to Regulate Neuronal Activity in the Mammalian Brain” from Patrick Aldrin-Kirk and Tomas Björklund (DOI: 10.1007/978-1-4939-9065-8_4).

For this particular study, Gq-coupled hM3D DREADD fused with mCherry, and Gi-coupled hM4D DREADD fused with mCherry, were chosen.

What is tetracycline-controlled transcriptional activation? This method allows precise, reversible and efficient spatiotemporal control of gene expression. It is often referred to as Tet-Off and Tet-On gene expression.

In the Tet-Off system, gene expression is turned off when tetracycline – or one of its derivatives, such as doxycycline – is administered. Conversely, the Tet-On system allows the gene expression by the addition of doxycycline. Therefore, one can control the expression of a gene of interest in freely behaving animals – by regulating their ingestion of doxycycline in water or food.

Why use tetracycline-controlled transcriptional activation? In this project, further study of the involvement of MCH neurons in memory mechanisms was assessed in mice. The team compared the performances at memory tasks with MCH mRNA-expressing neurons either present or ablated. They could hereby determine the role of MCH neurons in various types of memories.

What technology does MCI recommend? If your gene of interest should be active and only be turned off occasionally, the Tet-Off system will be the most appropriate method for your experiment.

In contrast, if your gene of interest should be inactive and only turned on occasionally, the Tet-On system will be the method to opt for.

Several supplierscan provide you with Tet systems products and services, for example plasmids or transgenic animals. We recommend you to visit theTet System website for more information.

What is optogenetic manipulation? Optogenetics is a technique that allows the manipulation of neuronal activity. This method allows for targeted excitation and/or inhibition of specific neuronal populations.

Similar to chemogenetics, a genetic strategy is employed to express a particular protein of interest in the targeted cells. This protein – called the optogenetic actuator – has the unique characteristic to be a light-sensitive ion channel. When illuminated with the corresponding wavelength, the channel allows flow of ions through the cell membrane. This method offers spatiotemporal control of neuronal excitability in living tissue.

Why use optogenetics? Optogenetics is a powerful method for targeted control of single-cell or neuronal populations. Unlike chemogenetics, it doesn’t require the administration of a synthetic drug. It offers temporal resolution to rapidly excite or inhibit neurons. Coupled with powerful illuminators such as the Polygon1000 by Mightex Systems, it allows subcellular resolution stimulation. Therefore, this method is extremely popular to study neuronal networks, both in vivo and in vitro.

For this project, channelrhodopsin2 (ChR2) was expressed in MCH neurons. Light stimulation of ChR2 in freely moving animals allowed to determine which memory types were impaired by the MCH neurons activity.

Secondly, to determine state dependent activity of MCH neurons, the optogenetic silencer Archaerhodopsin-T (ArchT) was expressed in MCH neurons. Inhibition of MCH neurons during REM sleep, non-REM sleep and wakefulness showed that only one subpopulation neurons – see paragraph 6 In vivo single-cell calcium imaging in freely moving animals – were related to memory impairment.

What technology does MCI recommend? For use in freely behaving animals, optogenetics require the implant of an optical fiber – to provide the illumination necessary to control the neuronal activity. With most available solutions, the optical fiber is connected to a stimulation unit that provides the light. These tethered options involve some movement restrictions for the animal.

Therefore, we recommend the technology used by the team in this project. The Teleopto Wireless Optical Stimulation allows the animal complete freedom of movement. This equipment offers a wide range of options:

Single or dual illumination of the same area

Stimulation in 2 different locations, either simultaneously or independently

Different fiber diameters (500 μm for this project)

Great selection of LED wavelengths (470 nm for this project)

5. Fiber Photometry

What is fiber photometry? Fiber photometry is a method allowing the recording of fluorescent signals in the brain of freely moving animals. It is based on the principle of expressing a sensor (sometimes called optogenetic sensor) in targeted neuronal populations. The sensor emits a specific fluorescence when in presence of the chemical of interest, e.g. calcium or a neurotransmitter. Other sensors further report on the vesicular release or the membrane voltage. Then, this difference in emitted fluorescence is detected by the system.

This combination with genetically encoded calcium indicators (GECIs) or genetically encoded voltage indicators (GEVIs) provides real-time information about the neurons of interest. Additionally, it can be associated with optogenetics actuators to simultaneously manipulate and report the activity of neuronal populations.

Why use fiber photometry? In this project, fiber photometry was used to collect the fluorescence from a GECI expressed in MCH neurons. This GECI is the GCaMP6; a fluorescent molecule that changes its fluorescence property in response to the binding of Ca2+ ions.

Therefore, changes in intracellular Ca2+ in MCH neurons were reported during the animal free behaviour. The team inserted fiber optics in the lateral hypothalamus to record the activity of MCH neurons.

What technology does MCI recommend? The tethered fiber photometry system used in this study delivered a blue excitation light – with 70 μW intensity at the tip of the fiber. Moreover, the fiber had a numerical aperture of 0.39 and a diameter of 400 μm.

As no optogenetic stimulation was required, we can also recommend a completely wireless solution – the TeleFipho for Wireless Fiber Photometry. This offers similar fiber characteristics with adjustable light excitation power between 10 and 300 μW, N.A. of 0.39, and a 400 μm fiber diameter. Furthermore, this option also solves all the issues related to tethered experiments – such as movement restrictions and recording artefacts – through a clever design of placing the system components onto the headstage.

6. In vivo single-cell calcium imaging in freely moving animals

What is in vivo single-cell calcium imaging in freely moving animals? On the same basis as fiber photometry, calcium imaging allows the monitoring of neuronal population activity in freely behaving animals. However, it provides the user with the possibility to gain spatial information. Thus, the system includes more complex optical components, allowing for visualisation of single-cell Ca2+ status.

Why use in vivo single-cell calcium imaging? In this project, the team investigated the activity of MCH neurons during different arousal states. However, fiber photometry only detected the population activity. Using single-cell calcium imaging allowed the team to measure the activity of a single MCH neuron. These individual recordings demonstrated the presence of several subpopulations of MCH neurons. These subpopulations have different roles depending on the wakefulness or sleep phase.

The OASIS Implant enables the user to do imaging and patterned optical stimulation at any depth in brain tissue of behaving animals.

What technology does MCI recommend? For this experiment, the team used the miniscope system. This technology includes all the optical components in a bulky headstage. In this way, the animal doesn’t have an optical fiber connecting it to a main unit. However, the system isn’t completely wireless, because – the animal is tethered to the collecting unit by the data transferring wire.

TheOASIS Implant by Mightex Systems is a ground-breaking optical fiberscope for simultaneous illumination and imaging of neuronal networks. It can be used in the deep-brain, cortex and multiple other brain or spinal cord regions. Admittedly, the animal is connected to the main unit by a flexible imaging fiber. Nonetheless, it gives this system the opportunity to develop its capacities beyond the limitations of the miniscopes. The OASIS Implant provides the net advantage for behavioural assays of using a compact and light headmount (as little as 0.7g, which is up to 3X lighter than miniscope alternatives). It also offers other key benefits:

7. EEG and EMG

What are EEG and EMG? Electroencephalography (EEG) is an electrophysiological method to measure gross electrical activity at the surface of the brain. It allows for detection of a sum of electrical events with precise time resolution. EEG is a non-invasive technique for clinical research. However, it requires implantation of the recording device for freely moving animals, adding a surgical procedure to the experimental protocol.

Electromyography (EMG) is also an electrophysiological technique, allowing for measurements of electrical activity in muscles.

Why use EEG and EMG? Combining recordings of EEG and EMG allow for the experimenter to determine the vigilance state of the subject. In this project, the team was interested in determining the different arousal and wakefulness phases of the animals. During the non-rapid eye movement (NREM) sleep phase, the EEG records a synchronization of slow-wave electrical activity. In contrast, high-frequency and low-amplitude activity is recorded during both rapid-eye movement (REM) sleep and wakefulness. In parallel, the EMG monitors the increase in activity during wakefulness in comparison to sleep. Therefore, these two methods combined determine with precision the transition from one state to another.

8. Electrophysiological recordings in brain slices

What are brain slice electrophysiological recordings? Electrophysiology is the study of the electrical properties of biological cells and tissues. In neuroscience, it includes measuring the electrical activity of neurons. For this, a brain extraction procedure is performed, during which slices are prepared. They are kept in an incubation chamber containing a bath solution (aCSF), mimicking the composition of the cerebrospinal fluid. This method allows to maintain the neurons ex vivo for the remaining experimental time.

Several methods can be used to record the electrical activity of a single or group of neurons in these slices. All of which follow the approach of measuring the voltage or current changes within a cell.

Why use brain slice electrophysiological recordings? This method allows the user to study the electrical characteristics and changes of a single or group of neurons – while controlling the environment. One can modify the chemical composition or the temperature of the solution; the current or the voltage injected in the cell. Additionally, the experimenter can observe the neuronal response to a compound, an electrical pulse or the stimulation of optogenetic actuators.

Two specific techniques were used in this project:

Whole-cell patch clamp: a pipette – containing the electrode – is pressed against the cell membrane. A negative pressure is delivered to rupture the membrane, allowing the recording of intracellular electrical activity.

Loose cell-attached patch-clamp: a pipette with electrode is pressed against the cell membrane. However, the membrane isn’t ruptured, allowing to keep the cell intact to record action potentials.

In using this method, the researchers were able to complete in vivo data on several aspects. Loose cell-attached recordings were performed to confirm the chemogenetics inhibition of the MCH neurons. Secondly, the recordings were combined with optogenetic stimulation. Patch-clamp recordings of hippocampal pyramidal CA1 neurons were coupled with ChR2 stimulation in MCH nerve terminals. This demonstrated the impact of the MCH neurons on hippocampal neurons.

Additionally, we’ve launched a service to help researchers reaching their goals more effectively. The Bespoke Rig Design is intended for accompanying scientists in building of new electrophysiology and imaging workstations. The aim of this service is to explore in detail all the technical requisites. Our team then evaluates all available technologies in order to provide solutions specifically tailored to one’s custom requirements.

9. Calcium imaging in brain slices

What is calcium imaging in brain slices? The experimental protocol to prepare the brain slices is similar to that described in the electrophysiological recordings section above. Calcium (Ca2+) imaging has also been introduced in the in vivo single-cell Ca2+ imaging section. As in ex vivo electrophysiology, the main interest of brain slice calcium imaging is the complete control over the environment. Additionally, it provides access to tissue otherwise unreachable for in vivo recording.

Why use calcium imaging in brain slices? For this project, the researchers performed simultaneous Ca2+ imaging and electrophysiological recordings. This experiment showed the correlation between the action potential frequency and the increase in Ca2+ fluorescence intensity in MCH neurons. This depicted the action potential frequency required to induce a change in Ca2+ concentration.

What technology does MCI recommend? The team used the GCaMP6 as a Ca2+ indicator. Excitation light for GCaMP6 was provided by a blue LED through an optical fiber. The blue light (475 nm wavelength) had an output power of 9.7 mW through the fluorescence microscope objective lens.

MCI can recommend a wide range of LEDs available from Mightex Systems. There are loads of LED options to choose from, with different wavelengths and illumination power. We will gladly help you choose the bespoke light sources for your experiments.

What is qRT-PCR? This is a method aiming at studying the quantity of RNA in a sample. The RNA extracted from a sample is transcribed into complementary DNA (cDNA). This cDNA is then used for the qPCR analysis. This technique is fast and sensitive to analyse the gene expression and quantify the amount of RNA of interest.

Why use qRT-PCR? In this project, RNA was extracted from whole brains after the behavioural assays. The qRT-PCR analysis revealed pro-MCH mRNA amounts in animals with or without MCH neuron ablation.

What technology does MCI recommend? qRT-PCR is a routine technique in many labs. Therefore, there is a great selection of equipment available. However, for electrophysiologists performing single-cell patch-clamp, we recommend looking into the technique combining single-cell patch-clamp recordings and RT-PCR. This technique allows for the study of RNA expression in specifically selected and recorded neurons. We leave it to some of its technique “fathers” to explain the method in this fundamentals paper.

11. Histology

What is histology? Histology is the study of the anatomy of biological cells and tissues through a microscope. The sample – e.g. whole brain, brain slices…- is collected from the animal and fixated. Then, it is further sectioned or directly stained, allowing observation under a microscope.

Why use histology? Histology allows for the observation of cell and tissue structure. In this project, the team used c-Fos staining. c-Fos is often expressed when neurons fire action potentials. Therefore, c-Fos is widely used as a functional marker of activity in neurons.

Additional histology experiments allowed the team to confirm accurate expression of opsins in the neuronal population of interest.

What technology does MCI recommend? Preparation of the brain slices is a key factor in the success of histology experiments. Great consideration should be taken in deciding on the most suitable device for your research. If you would like to find out more about different options of vibratomes, microtomes, or cryostats, please get in touch.

Concerning the observation of the preparation, MCI developed its own widefield fluorescence microscope. The CleverScope provides the user with the ability to observe any kind of immunofluorescence staining. It is compatible with the whole range of LED wavelengths from our partner Mightex Systems.

Which methods would you combine in your research projects?

A multimethod approach isn’t a prerequisite to nicely designed projects. Some technologies are adapted to answer specific questions, leading to ground-breaking discoveries. However, combining several levels of study for a physiological question is interesting. Having the ability to measure the activity of single or populations of neurons in controlled conditions is complementary to identifying their responses in vivo.

In the past, complex projects involving expertise in varied domains – e.g. surgery, behaviour, electrophysiology, and imaging – used to require collaborations across departments and sometimes universities. Nowadays, an increasing number of departments, if not labs, have access to different layers of experimental protocols that reveal more defined characteristics in their data. The access to technology is broadening our perspective and unlocking more creative ways of undertaking research problems.

With the world of Neuroscience research at your feet, what will you endeavour next?